RNA interference (RNAi) is a post-transcriptional pathway in which double-stranded RNA (dsRNA) triggers the degradation of complementary mRNA in the cytoplasm of eukaryotic cells. In plants and in some animals, including Caenorhabditis elegans, initiation of RNAi in one cell can lead to sequence-specific RNA silencing in another cell, a phenomenon referred to as non-cell-autonomous RNAi. Until recently, this phenomenon had not been observed in mammalian cells. Here, we review emerging data demonstrating that non-cell-autonomous RNAi occurs in cultured mammalian cells. We discuss possible mechanisms for the transfer of RNAi between mammalian cells and highlight the implications of this phenomenon for the development of in vivo cell-based RNAi delivery.

Despite numerous reports of non-cell-autonomous RNAi in plants and in some animals, until recently, this phenomenon had not been observed in mammalian cells. Here, we review emerging data demonstrating the transfer of RNAi between cultured mammalian cells by both cell contact-independent and cell contact-dependent transfer mechanisms. The existence of non-cell-autonomous RNAi in mammalian cells has important implications for the development of in vivo cell-based RNAi delivery.

SID-1 (systemic RNAi-defective-1) is a protein channel that is necessary for the import of dsRNA into most cells in C. elegans (Winston et al, 2002; Feinberg and Hunter, 2003). Overexpression of the mammalian homologue of SID-1 (i.e., SIDT-1) was shown to increase internalization of siRNA and RNAi in mammalian cells soaked in siRNA-containing medium (Duxbury et al, 2005; Tsang et al, 2007) (Figure 2E). However, at endogenous levels, SIDT-1 expression appeared to be insufficient for siRNA uptake into mammalian cells (Tsang et al, 2007).

MECHANSIMS OF CELL CONTACT-DEPENDENT NON-CELL-AUTONOMOUS RNAi

Tunneling nanotubes

Tunneling nanotubes (TNTs) were first identified in cultured rat pheochromocytoma PC12 cells as intercellular structures with diameters of 50–200nm and lengths of up to several cell diameters (Rustom et al, 2004). They were shown to hover between cells, transferring membrane components and organelles between the cytosols of adjoining cells (Rustom et al, 2004). TNTs with a variety of diameters, lengths, and compositions have since been identified in vitro in numerous cell types (reviewed in Gerdes and Carvalho, 2008; Gurke et al, 2008) and in vivo between bone marrow-derived MHC class II-positive cells in the corneal stroma of mice (Chinnery et al, 2008).

Two types of TNTs have been identified in cultured human macrophages: TNTs less than 0.7μm in diameter that contain F-actin, and TNTs greater than 0.7μm in diameter that contain F-actin and microtubules (Önfelt et al, 2006). Bidirectional transfer of mitochondria and intracellular vesicles (including late endosomes and lysosomes) can occur in the latter variety of TNTs (Önfelt et al, 2006), suggesting that TNTs could mediate the transfer of miRNA-containing endosomal vesicles between cells (Figure 2F). In agreement, Belting and Wittrup (2008) reviewed TNTs as a potential mechanism for the intercellular transfer of genetic material, due in part to their ability to transfer endosomal vesicles between cells. Additionally, TNTs are remarkably similar to plasmodesmata (reviewed in Rustom, 2009), which are involved in the transfer of siRNAs between plant cells. In fact, donor human MSCs were shown to infuse siRNAs into co-cultured recipient neural progenitor cells (Mitchell et al, 2011; Olson et al, 2011) through TNTs and other mechanisms (Nolta JA, personal communication).

Gap junctions

Gap junctions are intercellular channels that span the plasma membranes of adjoining cells, connecting the cytosols and allowing for cell-to-cell communication (reviewed in Bruzzone et al, 1996). In a gap junction, each of two adjacent cells contains a connexon, which is a hemichannel composed of six connexin (Cx) proteins. Gap junctions form from two identical connexons (homotypic) or two different connexons (heterotypic). Most tissues express more than one type of connexin and connexins often have distinct tissue and cellular distributions. Gap junction intercellular communication (GJIC) allows water-soluble molecules (e.g., ions, second messengers, and small metabolites) to diffuse between cells (reviewed in Bruzzone et al, 1996). Emerging evidence suggests that GJIC also mediates the transfer of RNAi between cells.

An immunological synapse (IS) is a junction that forms at the interface of a T cell and an antigen-presenting cell (APC), allowing for cell-to-cell interactions to modulate the immune response (reviewed in Rodríguez-Fernández et al, 2010). Mittelbrunn et al (2011) demonstrated that miRNA-containing exosomes transferred from T cells to APCs upon antigen-induced IS formation, resulting in sequence-specific RNA silencing in the recipient APCs. The T cell MVBs polarized towards the IS, which enhanced the secretion of miRNA-containing exosomes (Mittelbrunn et al, 2011). These data suggest that RNAi may transfer between immune cells through ISs (Figure 2H).

CONSIDERATIONS IN STUDIES OF NON-CELL-AUTONOMOUS RNAi IN MAMMALIAN CELLS

The method of introducing the RNAi agent into the donor cells

Different methods of introducing the RNAi agent into the donor cells may impact the transfer of RNAi between cells. For example, a common method of transfecting cells with siRNA involves complexing the siRNA with a cationic lipid carrier to create siRNA-lipoplexes. Recent data suggest that a majority of siRNA-lipoplexes introduced into cells persist in endolysosomes (Lu et al, 2009). Because endolysosomal trafficking may be involved in intercellular transfer of RNAi (reviewed in Gibbings and Voinnet, 2010), siRNA-lipoplexes within endolysomes may be more likely to transfer between cells than siRNA introduced into cells by a different method.

The method of introducing the RNAi agent into the donor cells may also impact the potency of RNA silencing in the recipient cells. Assuming that the donor cells remain viable at the target site, donor cells stably expressing shRNA or miRNA may induce more potent RNA silencing in the recipient cells than donor cells transiently expressing siRNA.

Donor to recipient cell ratio

Increasing the ratio of siRNA-expressing donor cells to recipient cells in co-culture increased target gene knockdown in recipient cells (Valiunas et al, 2005; Wolvetang et al, 2007), suggesting that a higher ratio of donor to recipient cells results in more RNA silencing signals entering the recipient cells. By increasing the ratio of donor to recipient cells, we expect to eventually reach a maximum percentage of target gene knockdown in recipient cells (Figure 3). This maximum would depend on a variety of factors including the rate of target mRNA turnover in recipient cells, the number of RNA silencing signals transferred by each donor cell, and the potency of each RNA silencing signal. Additionally, in cell contact-dependent RNAi transfer, limited points of contact between the donor and recipient cells may increase the time of co-culture required to reach the maximum target gene knockdown in recipient cells or may reduce the maximum altogether.

RNAi in donor cells following intercellular transfer of RNAi to recipient cells

While the transfer of RNA silencing signals from donor to recipient cells may lessen the potency of RNAi in the donor cells, we predict that the transfer would not eliminate RNAi in the donor cells. Each activated RISC undergoes multiple rounds of RNA silencing (Hutvágner and Zamore, 2002), suggesting that potent RNA silencing could occur in the donor cells despite the transfer of some RNA silencing signals to the recipient cells. Additionally, an RNA-dependent RNA polymerase (RdRP) has recently been identified in mammalian cells (Maida et al, 2009).

RdRPs are primed by existing siRNAs to produce more dsRNAs, thereby amplifying RNA silencing (Sijen et al, 2001; reviewed in Nishikura, 2001). Thus, the transfer of RNA silencing signals from donor to recipient cells would not necessarily eliminate RNAi in the donor cells. Unfortunately, limited understanding of the molecular nature of the transferred RNA silencing signal makes it difficult to predict how RdRPs or the multi-turnover nature of RISC impacts the transfer of RNAi between mammalian cells.

It is unlikely that long dsRNA is the RNA silencing signal that transfers between mammalian cells, as dsRNAs longer than 30nt activate the interferon system (Minks et al, 1979; Manche et al, 1992). In agreement, Valiunas et al (2005), Wolvetang et al (2007), and Kizana et al (2009) hypothesized that Dicer-processed shRNA was the RNA silencing signal transferred between mammalian cells through gap junctions. Valiunas et al (2005) demonstrated that oligonucleotides (morphilinos) simulating siRNAs, with molecular weights of ∼2–4kDa, minor diameters of 1.0–1.1nm, and lengths of 7.6nm could diffuse between cells through C×43 gap junctions. The oligonucleotide permeation through the C×43 gap junctions decreased as the length of the oligonucleotides increased, which was expected as the minor diameter of the oligonucleotides was close to the pore diameter of the gap junctions (∼1.0–1.5nm) (Valiunas et al, 2005). A hybridized, double-stranded 12-mer oligonucleotide had significantly decreased permeation through the C×43 gap junctions when compared with a single-stranded 12-mer oligonucleotide, suggesting that single-stranded siRNAs are more likely to transfer through C×43 gap junctions than hybridized siRNAs (Valiunas et al, 2005). Additionally, Kizana et al (2009) determined that, compared with shRNA-expressing donor cells, co-cultured recipient cells contained 25% of the copy number of the antisense strand of shRNA, suggesting that the antisense strand of shRNA, or possibly the shRNA duplex, transfers from donor to recipient cells through C×43 gap junctions.

Rechavi et al (2009) demonstrated the transfer of 22-nt Cy3-labeled dsRNAs, or possibly metabolic products of those dsRNAs, from donor B cells to recipient T cells. However, 22-nt FITC-conjugated locked nucleic acids did not transfer between cells, suggesting that structural specificity is involved in intercellular RNA transfer (Rechavi et al, 2009). Additionally, Rechavi et al (2009) did not detect the movement of the RISC component Ago2 between donor and recipient cells, suggesting that small RNAs transfer between cells independently of Ago2. Based on these studies, we hypothesize that the antisense strand of Dicer-processed siRNA may be the RNA silencing signal that transfers between cells in non-cell-autonomous RNAi; however, we recognize that there may be different RNA silencing signals depending on the mechanism of non-cell-autonomous RNAi.

Endolysosmal trafficking and intercellular transfer of RNAi

There is increasing data linking RNAi to endolysosomal trafficking (reviewed in Siomi and Siomi, 2009; Gibbings and Voinnet, 2010). In the endolysosomal pathway, the endosomal sorting complex required for transport (ESCRT) is required for the invagination of the limiting endosomal membrane in MVBs to form ILVs (reviewed in Babst, 2005). ESCRT is also required for the sorting of endosomal cargo proteins into ILVs. MVBs can fuse with the lysosome for degradation or they can fuse with the plasma membrane to secrete their ILVs, which are then referred to as exosomes.

Despite the enormous potential of RNAi for disease therapy, current in vivo RNAi delivery strategies (reviewed in Whitehead et al, 2009; Lares et al, 2010), such as those using liposomes, cationic polymers, or viral vectors, have been hindered by a variety of challenges (reviewed in Trehan et al, 2010), including immune system activation and inefficient cell targeting. Cell-based RNAi delivery, in which donor cells act as RNAi delivery vehicles, can potentially overcome these challenges (reviewed in Brink et al, 2010; Brink et al, 2011). Autologous or immunoprivileged allogeneic donor cells may avoid host immune system activation, and certain cell types inherently migrate to tumors or wounds. Furthermore, in areas where traditional RNAi delivery vehicles have limited diffusion, the spread of RNAi may be more efficient by transferring the RNA silencing signal from cell to cell.

Emerging evidence demonstrates that non-cell-autonomous RNAi occurs in cultured mammalian cells. This phenomenon can occur by cell contact-independent mechanisms (Figure 2A-D), such as the shuttling of miRNA-containing MVs, exosomes, or apoptotic bodies between cells. Non-cell-autonomous RNAi can also occur by cell contact-dependent mechanisms, relying on TNTs, gap junctions, or ISs (Figure 2F-H). Non-cell-autonomous RNAi in mammalian cells may allow for the development of in vivo cell-based RNAi delivery, which has the potential to overcome major challenges in RNAi delivery and allow for effective RNAi therapies.

NotesSTATEMENT OF COMPETING INTERESTS

None declared

We would like to acknowledge Sally Griffith-Oh at the University of Wisconsin-Madison for help in creating Figure 2. This work was supported with start-up funds from the University of Wisconsin-Madison School of Pharmacy and through a Department of Defense (DoD) National Defense Science and Engineering Graduate (NDSEG) Fellowship to HC Cohen.

The RNAi pathway. Dicer cleaves long dsRNA in the form of pre-shRNA, pre-miRNA, or Dicer-substrate siRNA into ∼21–25-nt dsRNA duplexes (Hammond et al, 2000; Zamore et al, 2000), referred to as shRNAs, miRNAs, or siRNAs, respectively, to initiate RNAi. Most shRNAs and siRNAs have exact complementarity with their target mRNAs and result in mRNA cleavage by Ago2. Most miRNAs bind to the 3' untranslated region of their target mRNAs with imperfect complementarity, resulting in translational repression (reviewed in He and Hannon, 2004) or deadenylation and target mRNA degradation (reviewed in Fabian et al, 2010). Alternatively, ∼21–25nt siRNAs can be directly transfected into the cytoplasm of cells to initiate RNAi. Note that the term siRNA can also be used more generally to describe any of the forms of ∼21–25nt dsRNA duplexes that initiate RNAi.

[Figure ID: F2]

Figure 2.

Possible mechanisms of non-cell-autonomous RNAi in cultured mammalian cells. Non-cell-autonomous RNAi can occur by cell contact-independent mechanisms, such as the shuttling of miRNA-containing MVs (A), exosomes (B), or apoptotic bodies (C) between cells. Additionally, miRNA-Ago2 complexes may induce RNAi in recipient cells (D). siRNAs and miRNAs may also utilize SIDT-1, when present at high expression levels, for entry into cells and to generate RNAi (E). Non-cell-autonomous RNAi can also occur by cell contact-dependent mechanisms, relying on TNTs (F), gap junctions (G), or ISs (H).

[Figure ID: F3]

Figure 3.

The predicted transfer of RNAi when the ratio of siRNA-expressing donor cells to recipient cells in co-culture is increased. Valiunas et al (2005) observed a greater percentage of target gene knockdown in recipient cells when the ratio of donor to recipient cells increased from 1:1 to 2:1. Similarly, Wolvetang et al (2007) observed a progressive increase in target gene knockdown in recipient cells when the ratio of donor to recipient cells increased from 1:1 to 2:1 to 3:1. By increasing the ratio of donor to recipient cells, we expect to eventually reach a maximum percentage of target gene knockdown in recipient cells. In cell contact-dependent transfer of RNAi, such as by GJIC, limited points of contact between the donor and recipient cells may reduce the maximum target gene knockdown in recipient cells.